DSpace Collection:http://hdl.handle.net/2381/382018-02-18T05:08:56Z2018-02-18T05:08:56ZThe cytoskeleton adaptor protein ankyrin-1 is upregulated by p53 following DNA damage and alters cell migrationHall, A. E.Lu, W. T.Godfrey, J. D.Antonov, A. V.Paicu, C.Moxon, S.Dalmay, T.Wilczynska, A.Muller, P. A.Bushell, Martin D.http://hdl.handle.net/2381/381422016-10-06T02:18:45Z2016-10-05T10:07:21ZTitle: The cytoskeleton adaptor protein ankyrin-1 is upregulated by p53 following DNA damage and alters cell migration
Authors: Hall, A. E.; Lu, W. T.; Godfrey, J. D.; Antonov, A. V.; Paicu, C.; Moxon, S.; Dalmay, T.; Wilczynska, A.; Muller, P. A.; Bushell, Martin D.
Abstract: The integrity of the genome is maintained by a host of surveillance and repair mechanisms that are pivotal for cellular function. The tumour suppressor protein p53 is a major component of the DNA damage response pathway and plays a vital role in the maintenance of cell-cycle checkpoints. Here we show that a microRNA, miR-486, and its host gene ankyrin-1 (ANK1) are induced by p53 following DNA damage. Strikingly, the cytoskeleton adaptor protein ankyrin-1 was induced over 80-fold following DNA damage. ANK1 is upregulated in response to a variety of DNA damage agents in a range of cell types. We demonstrate that miR-486-5p is involved in controlling G1/S transition following DNA damage, whereas the induction of the ankyrin-1 protein alters the structure of the actin cytoskeleton and sustains limited cell migration during DNA damage. Importantly, we found that higher ANK1 expression correlates with decreased survival in cancer patients. Thus, these observations highlight ANK1 as an important effector downstream of the p53 pathway.2016-10-05T10:07:21Zp73 promotes glioblastoma cell invasion by directly activating POSTN (periostin) expressionLandré, VivienAntonov, AlexeyKnight, RichardMelino, Gerryhttp://hdl.handle.net/2381/377952016-06-21T02:16:59Z2016-06-20T13:44:33ZTitle: p73 promotes glioblastoma cell invasion by directly activating POSTN (periostin) expression
Authors: Landré, Vivien; Antonov, Alexey; Knight, Richard; Melino, Gerry
Abstract: Glioblastoma Multiforme is one of the most highly metastatic cancers and constitutes 70% of all gliomas. Despite aggressive treatments these tumours have an exceptionally bad prognosis, mainly due to therapy resistance and tumour recurrence. Here we show that the transcription factor p73 confers an invasive phenotype by directly activating expression of POSTN (periostin, HGNC:16953) in glioblastoma cells. Knock down of endogenous p73 reduces invasiveness and chemo-resistance, and promotes differentiation in vitro. Using chromatin immunoprecipitation and reporter assays we demonstrate that POSTN, an integrin binding protein that has recently been shown to play a major role in metastasis, is a transcriptional target of TAp73. We further show that POSTN overexpression is sufficient to rescue the invasive phenotype of glioblastoma cells after p73 knock down. Additionally, bioinformatics analysis revealed that an intact p73/ POSTN axis, where POSTN and p73 expression is correlated, predicts bad prognosis in several cancer types. Taken together, our results support a novel role of TAp73 in controlling glioblastoma cell invasion by regulating the expression of the matricellular protein POSTN.2016-06-20T13:44:33ZExpression and activity of eIF6 trigger Malignant Pleural Mesothelioma growth in vivoMiluzio, A.Oliveto, S.Pesce, E.Mutti, L.Murer, B.Grosso, StefanoRicciardi, S.Brina, D.Biffo, S.http://hdl.handle.net/2381/377942016-06-21T02:17:14Z2016-06-20T13:22:12ZTitle: Expression and activity of eIF6 trigger Malignant Pleural Mesothelioma growth in vivo
Authors: Miluzio, A.; Oliveto, S.; Pesce, E.; Mutti, L.; Murer, B.; Grosso, Stefano; Ricciardi, S.; Brina, D.; Biffo, S.
Abstract: eIF6 is an antiassociation factor that regulates the availability of active 80S. Its activation is driven by the RACK1/PKCβ axis, in a mTORc1 independent manner. We previously described that eIF6 haploinsufficiency causes a striking survival in the Eμ-Myc mouse lymphoma model, with lifespans extended up to 18 months. Here we screen for eIF6 expression in human cancers. We show that Malignant Pleural Mesothelioma tumors (MPM) and a MPM cell line (REN cells) contain high levels of hyperphosphorylated eIF6. Enzastaurin is a PKC beta inhibitor used in clinical trials. We prove that Enzastaurin treatment decreases eIF6 phosphorylation rate, but not eIF6 protein stability. The growth of REN, in vivo, and metastasis are reduced by either Enzastaurin treatment or eIF6 shRNA. Molecular analysis reveals that eIF6 manipulation affects the metabolic status of malignant mesothelioma cells. Less glycolysis and less ATP content are evident in REN cells depleted for eIF6 or treated with Enzastaurin (Anti-Warburg effect). We propose that eIF6 is necessary for malignant mesothelioma growth, in vivo, and can be targeted by kinase inhibitors.2016-06-20T13:22:12ZThe anti-HER3 (ErbB3) therapeutic antibody 9F7-F11 induces HER3 ubiquitination and degradation in tumors through JNK1/2- dependent ITCH/AIP4 activationLe Clorennec, C.Lazrek, Y.Dubreuil, O.Larbouret, C.Poul, M-A.Mondon, P.Melino, GerryPèlegrin, A.Chardès, T.http://hdl.handle.net/2381/377932016-06-21T02:17:07Z2016-06-20T10:19:05ZTitle: The anti-HER3 (ErbB3) therapeutic antibody 9F7-F11 induces HER3 ubiquitination and degradation in tumors through JNK1/2- dependent ITCH/AIP4 activation
Authors: Le Clorennec, C.; Lazrek, Y.; Dubreuil, O.; Larbouret, C.; Poul, M-A.; Mondon, P.; Melino, Gerry; Pèlegrin, A.; Chardès, T.
Abstract: We characterized the mechanism of action of the neuregulin-non-competitive anti-HER3 therapeutic antibody 9F7-F11 that blocks the PI3K/AKT pathway, leading to cell cycle arrest and apoptosis in vitro and regression of pancreatic and breast cancer in vivo. We found that 9F7-F11 induces rapid HER3 down-regulation. Specifically, 9F7-F11-induced HER3 ubiquitination and degradation in pancreatic, breast and prostate cancer cell lines was driven mainly by the itchy E3 ubiquitin ligase (ITCH/AIP4). Overexpression of the ITCH/AIP4 inhibitor N4BP1 or small-interfering RNA-mediated knockdown of ITCH/AIP4 inhibited HER3 ubiquitination/degradation and PI3K/AKT signaling blockade induced by 9F7-F11. Moreover, 9F7-F11-mediated JNK1/2 phosphorylation led to ITCH/AIP4 activation and recruitment to HER3 for receptor ubiquitination and degradation. ITCH/AIP4 activity was activated by the deubiquitinases USP8 and USP9X, as demonstrated by RNA interference. Taken together, our results suggest that 9F7-F11-induced HER3 ubiquitination and degradation in cancer cells mainly occurs through JNK1/2-dependent ITCH/AIP4 activation.2016-06-20T10:19:05ZTemporal Regulation of Distinct Internal Ribosome Entry Sites of the Dicistroviridae Cricket Paralysis VirusKhong, AnthonyBonderoff, Jennifer M.Spriggs, Ruth V.Tammpere, ErikKerr, Craig H.Jackson, Thomas J.Willis, Anne E.Jan, Erichttp://hdl.handle.net/2381/376792016-06-02T02:14:13Z2016-06-01T13:11:47ZTitle: Temporal Regulation of Distinct Internal Ribosome Entry Sites of the Dicistroviridae Cricket Paralysis Virus
Authors: Khong, Anthony; Bonderoff, Jennifer M.; Spriggs, Ruth V.; Tammpere, Erik; Kerr, Craig H.; Jackson, Thomas J.; Willis, Anne E.; Jan, Eric
Abstract: Internal ribosome entry is a key mechanism for viral protein synthesis in a subset of RNA viruses. Cricket paralysis virus (CrPV), a member of Dicistroviridae, has a positive-sense single strand RNA genome that contains two internal ribosome entry sites (IRES), a 5'untranslated region (5'UTR) and intergenic region (IGR) IRES, that direct translation of open reading frames (ORF) encoding the viral non-structural and structural proteins, respectively. The regulation of and the significance of the CrPV IRESs during infection are not fully understood. In this study, using a series of biochemical assays including radioactive-pulse labelling, reporter RNA assays and ribosome profiling, we demonstrate that while 5'UTR IRES translational activity is constant throughout infection, IGR IRES translation is delayed and then stimulated two to three hours post infection. The delay in IGR IRES translation is not affected by inhibiting global translation prematurely via treatment with Pateamine A. Using a CrPV replicon that uncouples viral translation and replication, we show that the increase in IGR IRES translation is dependent on expression of non-structural proteins and is greatly stimulated when replication is active. Temporal regulation by distinct IRESs within the CrPV genome is an effective viral strategy to ensure optimal timing and expression of viral proteins to facilitate infection.2016-06-01T13:11:47ZP53 functional abnormality in mesenchymal stem cells promotes osteosarcoma developmentVelletri, T.Xie, N.Wang, Y.Huang, Y.Yang, Q.Chen, X.Chen, Q.Shou, P.Gan, Y.Cao, G.Melino, G.Shi, Y.http://hdl.handle.net/2381/374452016-04-26T02:05:11Z2016-04-25T15:58:00ZTitle: P53 functional abnormality in mesenchymal stem cells promotes osteosarcoma development
Authors: Velletri, T.; Xie, N.; Wang, Y.; Huang, Y.; Yang, Q.; Chen, X.; Chen, Q.; Shou, P.; Gan, Y.; Cao, G.; Melino, G.; Shi, Y.
Abstract: It has been shown that p53 has a critical role in the differentiation and functionality of various multipotent progenitor cells. P53 mutations can lead to genome instability and subsequent functional alterations and aberrant transformation of mesenchymal stem cells (MSCs). The significance of p53 in safeguarding our body from developing osteosarcoma (OS) is well recognized. During bone remodeling, p53 has a key role in negatively regulating key factors orchestrating the early stages of osteogenic differentiation of MSCs. Interestingly, changes in the p53 status can compromise bone homeostasis and affect the tumor microenvironment. This review aims to provide a unique opportunity to study the p53 function in MSCs and OS. In the context of loss of function of p53, we provide a model for two sources of OS: MSCs as progenitor cells of osteoblasts and bone tumor microenvironment components. Standing at the bone remodeling point of view, in this review we will first explain the determinant function of p53 in OS development. We will then summarize the role of p53 in monitoring MSC fidelity and in regulating MSC differentiation programs during osteogenesis. Finally, we will discuss the importance of loss of p53 function in tissue microenvironment. We expect that the information provided herein could lead to better understanding and treatment of OS.2016-04-25T15:58:00ZEpithelial transglutaminase 2 is needed for T cell interleukin-17 production and subsequent pulmonary inflammation and fibrosis in bleomycin-treated miceOh, K.Park, H-B.Byoun, O-J.Shin, D-M.Jeong, E. M.Kim, Y. W.Kim, Y. S.Melino, GerryKim, I-G.Lee, D-S.http://hdl.handle.net/2381/374302016-04-23T02:32:32Z2016-04-22T09:21:30ZTitle: Epithelial transglutaminase 2 is needed for T cell interleukin-17 production and subsequent pulmonary inflammation and fibrosis in bleomycin-treated mice
Authors: Oh, K.; Park, H-B.; Byoun, O-J.; Shin, D-M.; Jeong, E. M.; Kim, Y. W.; Kim, Y. S.; Melino, Gerry; Kim, I-G.; Lee, D-S.
Abstract: Pulmonary fibrosis is a potentially life-threatening disease that may be caused by overt or asymptomatic inflammatory responses. However, the precise mechanisms by which tissue injury is translated into inflammation and consequent fibrosis remain to be established. Here, we show that in a lung injury model, bleomycin induced the secretion of IL-6 by epithelial cells in a transglutaminase 2 (TG2)–dependent manner. This response represents a key step in the differentiation of IL-17–producing T cells and subsequent inflammatory amplification in the lung. The essential role of epithelial cells, but not inflammatory cells, TG2 was confirmed in bone marrow chimeras; chimeras made in TG2-deficient recipients showed reduced inflammation and fibrosis, compared with those in wild-type mice, regardless of the bone marrow cell phenotype. Epithelial TG2 thus appears to be a critical inducer of inflammation after noninfectious pulmonary injury. We further demonstrated that fibroblast-derived TG2, acting downstream of transforming growth factor-β, is also important in the effector phase of fibrogenesis. Therefore, TG2 represents an interesting potential target for therapeutic intervention.
Description: Supplemental Material can be found at: http://jem.rupress.org/content/suppl/2011/07/10/jem.20101457.DC1.html2016-04-22T09:21:30ZKnockdown of Hsc70-5/mortalin induces loss of synaptic mitochondria in a Drosophila Parkinson's disease modelZhu, J. Y.Vereshchagina, N.Sreekumar, V.Burbulla, L. F.Costa, A. C.Daub, K. J.Woitalla, D.Martins, L. MiguelKrüger, R.Rasse, T. M.http://hdl.handle.net/2381/374222016-07-13T14:11:31Z2016-04-21T10:01:23ZTitle: Knockdown of Hsc70-5/mortalin induces loss of synaptic mitochondria in a Drosophila Parkinson's disease model
Authors: Zhu, J. Y.; Vereshchagina, N.; Sreekumar, V.; Burbulla, L. F.; Costa, A. C.; Daub, K. J.; Woitalla, D.; Martins, L. Miguel; Krüger, R.; Rasse, T. M.
Abstract: Mortalin is an essential component of the molecular machinery that imports nuclear-encoded proteins into mitochondria, assists in their folding, and protects against damage upon accumulation of dysfunctional, unfolded proteins in aging mitochondria. Mortalin dysfunction associated with Parkinson's disease (PD) increases the vulnerability of cultured cells to proteolytic stress and leads to changes in mitochondrial function and morphology. To date, Drosophila melanogaster has been successfully used to investigate pathogenesis following the loss of several other PD-associated genes. We generated the first loss-of-Hsc70-5/mortalin-function Drosophila model. The reduction of Mortalin expression recapitulates some of the defects observed in the existing Drosophila PD-models, which include reduced ATP levels, abnormal wing posture, shortened life span, and reduced spontaneous locomotor and climbing ability. Dopaminergic neurons seem to be more sensitive to the loss of mortalin than other neuronal sub-types and non-neuronal tissues. The loss of synaptic mitochondria is an early pathological change that might cause later degenerative events. It precedes both behavioral abnormalities and structural changes at the neuromuscular junction (NMJ) of mortalin-knockdown larvae that exhibit increased mitochondrial fragmentation. Autophagy is concomitantly up-regulated, suggesting that mitochondria are degraded via mitophagy. Ex vivo data from human fibroblasts identifies increased mitophagy as an early pathological change that precedes apoptosis. Given the specificity of the observed defects, we are confident that the loss-of-mortalin model presented in this study will be useful for further dissection of the complex network of pathways that underlie the development of mitochondrial parkinsonism.2016-04-21T10:01:23ZIdebenone and resveratrol extend lifespan and improve motor function of HtrA2 knockout miceGerhardt, E.Gräber, S.Szego, E. M.Moisoi, N.Martins, L. MiguelOuteiro, T. F.Kermer, P.http://hdl.handle.net/2381/374212016-07-13T14:11:19Z2016-04-21T09:59:37ZTitle: Idebenone and resveratrol extend lifespan and improve motor function of HtrA2 knockout mice
Authors: Gerhardt, E.; Gräber, S.; Szego, E. M.; Moisoi, N.; Martins, L. Miguel; Outeiro, T. F.; Kermer, P.
Abstract: Heterozygous loss-of-function mutation of the human gene for the mitochondrial protease HtrA2 has been associated with increased risk to develop mitochondrial dysfunction, a process known to contribute to neurodegenerative disorders such as Huntington's disease (HD) and Parkinson's disease (PD). Knockout of HtrA2 in mice also leads to mitochondrial dysfunction and to phenotypes that resemble those found in neurodegenerative disorders and, ultimately, lead to death of animals around postnatal day 30. Here, we show that Idebenone, a synthetic antioxidant of the coenzyme Q family, and Resveratrol, a bioactive compound extracted from grapes, are both able to ameliorate this phenotype. Feeding HtrA2 knockout mice with either compound extends lifespan and delays worsening of the motor phenotype. Experiments conducted in cell culture and on brain tissue of mice revealed that each compound has a different mechanism of action. While Idebenone acts by downregulating the integrated stress response, Resveratrol acts by attenuating apoptosis at the level of Bax. These activities can account for the delay in neuronal degeneration in the striata of these mice and illustrate the potential of these compounds as effective therapeutic approaches against neurodegenerative disorders such as HD or PD.2016-04-21T09:59:37ZCancer and Neurodegeneration: Between the Devil and the Deep Blue SeaPlun-Favreau, H.Lewis, P. A.Hardy, J.Martins, L. MiguelWood, N. W.http://hdl.handle.net/2381/374182016-07-13T14:10:51Z2016-04-21T09:52:56ZTitle: Cancer and Neurodegeneration: Between the Devil and the Deep Blue Sea
Authors: Plun-Favreau, H.; Lewis, P. A.; Hardy, J.; Martins, L. Miguel; Wood, N. W.
Abstract: Cancer and neurodegeneration are often thought of as disease mechanisms at opposite ends of a spectrum; one due to enhanced resistance to cell death and the other due to premature cell death. There is now accumulating evidence to link these two disparate processes. An increasing number of genetic studies add weight to epidemiological evidence suggesting that sufferers of a neurodegenerative disorder have a reduced incidence for most cancers, but an increased risk for other cancers. Many of the genes associated with either cancer and/or neurodegeneration play a central role in cell cycle control, DNA repair, and kinase signalling. However, the links between these two families of diseases remain to be proven. In this review, we discuss recent and sometimes as yet incomplete genetic discoveries that highlight the overlap of molecular pathways implicated in cancer and neurodegeneration.2016-04-21T09:52:56ZGenetic analysis of mitochondrial protein misfolding in Drosophila melanogasterPimenta de Castro, I.Costa, A. C.Lam, D.Tufi, R.Fedele, V.Moisoi, N.Dinsdale, D.Deas, E.Loh, S. H.Martins, L. Miguelhttp://hdl.handle.net/2381/374042016-07-13T14:11:09Z2016-04-20T10:52:46ZTitle: Genetic analysis of mitochondrial protein misfolding in Drosophila melanogaster
Authors: Pimenta de Castro, I.; Costa, A. C.; Lam, D.; Tufi, R.; Fedele, V.; Moisoi, N.; Dinsdale, D.; Deas, E.; Loh, S. H.; Martins, L. Miguel
Abstract: Protein misfolding has a key role in several neurological disorders including Parkinson's disease. Although a clear mechanism for such proteinopathic diseases is well established when aggregated proteins accumulate in the cytosol, cell nucleus, endoplasmic reticulum and extracellular space, little is known about the role of protein aggregation in the mitochondria. Here we show that mutations in both human and fly PINK1 result in higher levels of misfolded components of respiratory complexes and increase in markers of the mitochondrial unfolded protein response. Through the development of a genetic model of mitochondrial protein misfolding employing Drosophila melanogaster, we show that the in vivo accumulation of an unfolded protein in mitochondria results in the activation of AMP-activated protein kinase-dependent autophagy and phenocopies of pink1 and parkin mutants. Parkin expression acts to clear mitochondria with enhanced levels of misfolded proteins by promoting their autophagic degradation in vivo, and refractory to Sigma P (ref(2)P), the Drosophila orthologue of mammalian p62, is a critical downstream effector of this quality control pathway. We show that in flies, a pathway involving pink1, parkin and ref(2)P has a role in the maintenance of a viable pool of cellular mitochondria by promoting organellar quality control.2016-04-20T10:52:46ZA Unique Protein Phosphatase with Kelch-Like Domains (PPKL) in Plasmodium Modulates Ookinete Differentiation, Motility and InvasionGuttery, D. S.Poulin, B.Ferguson, D. J. P.Szoeor, B.Wickstead, B.Carroll, P. L.Ramakrishnan, C.Brady, D.Patzewitz, E-M.Straschil, U.Solyakov, L.Green, J. L.Sinden, R. E.Tobin, Andrew B.Holder, A. A.Tewari, R.http://hdl.handle.net/2381/373482016-04-19T02:15:57Z2016-04-18T12:04:15ZTitle: A Unique Protein Phosphatase with Kelch-Like Domains (PPKL) in Plasmodium Modulates Ookinete Differentiation, Motility and Invasion
Authors: Guttery, D. S.; Poulin, B.; Ferguson, D. J. P.; Szoeor, B.; Wickstead, B.; Carroll, P. L.; Ramakrishnan, C.; Brady, D.; Patzewitz, E-M.; Straschil, U.; Solyakov, L.; Green, J. L.; Sinden, R. E.; Tobin, Andrew B.; Holder, A. A.; Tewari, R.
Abstract: Protein phosphorylation and dephosphorylation (catalysed by kinases and phosphatases, respectively) are post-translational modifications that play key roles in many eukaryotic signalling pathways, and are often deregulated in a number of pathological conditions in humans. In the malaria parasite Plasmodium, functional insights into its kinome have only recently been achieved, with over half being essential for blood stage development and another 14 kinases being essential for sexual development and mosquito transmission. However, functions for any of the plasmodial protein phosphatases are unknown. Here, we use reverse genetics in the rodent malaria model, Plasmodium berghei, to examine the role of a unique protein phosphatase containing kelch-like domains (termed PPKL) from a family related to Arabidopsis BSU1. Phylogenetic analysis confirmed that the family of BSU1-like proteins including PPKL is encoded in the genomes of land plants, green algae and alveolates, but not in other eukaryotic lineages. Furthermore, PPKL was observed in a distinct family, separate to the most closely-related phosphatase family, PP1. In our genetic approach, C-terminal GFP fusion with PPKL showed an active protein phosphatase preferentially expressed in female gametocytes and ookinetes. Deletion of the endogenous ppkl gene caused abnormal ookinete development and differentiation, and dissociated apical microtubules from the inner-membrane complex, generating an immotile phenotype and failure to invade the mosquito mid-gut epithelium. These observations were substantiated by changes in localisation of cytoskeletal tubulin and actin, and the micronemal protein CTRP in the knockout mutant as assessed by indirect immunofluorescence. Finally, increased mRNA expression of dozi, a RNA helicase vital to zygote development was observed in ppkl− mutants, with global phosphorylation studies of ookinete differentiation from 1.5–24 h post-fertilisation indicating major changes in the first hours of zygote development. Our work demonstrates a stage-specific essentiality of the unique PPKL enzyme, which modulates parasite differentiation, motility and transmission.2016-04-18T12:04:15ZDrosophila ref(2)P is required for the parkin-mediated suppression of mitochondrial dysfunction in pink1 mutantsde Castro, I. P.Costa, A. C.Celardo, I.Tufi, R.Dinsdale, D.Loh, S. H.Martins, L. Miguelhttp://hdl.handle.net/2381/373442016-07-13T14:11:00Z2016-04-18T11:52:14ZTitle: Drosophila ref(2)P is required for the parkin-mediated suppression of mitochondrial dysfunction in pink1 mutants
Authors: de Castro, I. P.; Costa, A. C.; Celardo, I.; Tufi, R.; Dinsdale, D.; Loh, S. H.; Martins, L. Miguel
Abstract: Autophagy is a critical regulator of organellar homeostasis, particularly of mitochondria. Upon the loss of membrane potential, dysfunctional mitochondria are selectively removed by autophagy through recruitment of the E3 ligase Parkin by the PTEN-induced kinase 1 (PINK1) and subsequent ubiquitination of mitochondrial membrane proteins. Mammalian sequestrome-1 (p62/SQSTM1) is an autophagy adaptor, which has been proposed to shuttle ubiquitinated cargo for autophagic degradation downstream of Parkin. Here, we show that loss of ref(2)P, the Drosophila orthologue of mammalian P62, results in abnormalities, including mitochondrial defects and an accumulation of mitochondrial DNA with heteroplasmic mutations, correlated with locomotor defects. Furthermore, we show that expression of Ref(2)P is able to ameliorate the defects caused by loss of Pink1 and that this depends on the presence of functional Parkin. Finally, we show that both the PB1 and UBA domains of Ref(2)P are crucial for mitochondrial clustering. We conclude that Ref(2)P is a crucial downstream effector of a pathway involving Pink1 and Parkin and is responsible for the maintenance of a viable pool of cellular mitochondria by promoting their aggregation and autophagic clearance.2016-04-18T11:52:14ZDistinct phosphorylation sites on the ghrelin receptor, GHSR1a, establish a code that determines the functions of ß-arrestinsBouzo-Lorenzo, M.Santo-Zas, I.Lodeiro, M.Nogueiras, R.Casanueva, F. F.Castro, M.Pazos, Y.Tobin, Andrew B.Butcher, A. J.Camiña, J. P.http://hdl.handle.net/2381/373402016-04-19T02:04:37Z2016-04-18T11:32:29ZTitle: Distinct phosphorylation sites on the ghrelin receptor, GHSR1a, establish a code that determines the functions of ß-arrestins
Authors: Bouzo-Lorenzo, M.; Santo-Zas, I.; Lodeiro, M.; Nogueiras, R.; Casanueva, F. F.; Castro, M.; Pazos, Y.; Tobin, Andrew B.; Butcher, A. J.; Camiña, J. P.
Abstract: The growth hormone secretagogue receptor, GHSR1a, mediates the biological activities of ghrelin, which includes the secretion of growth hormone, as well as the stimulation of appetite, food intake and maintenance of energy homeostasis. Mapping phosphorylation sites on GHSR1a and knowledge of how these sites control specific functional consequences unlocks new strategies for the development of therapeutic agents targeting individual functions. Herein, we have identified the phosphorylation of different sets of sites within GHSR1a which engender distinct functionality of ß-arrestins. More specifically, the Ser(362), Ser(363) and Thr(366) residues at the carboxyl-terminal tail were primarily responsible for ß-arrestin 1 and 2 binding, internalization and ß-arrestin-mediated proliferation and adipogenesis. The Thr(350) and Ser(349) are not necessary for ß-arrestin recruitment, but are involved in the stabilization of the GHSR1a-ß-arrestin complex in a manner that determines the ultimate cellular consequences of ß-arrestin signaling. We further demonstrated that the mitogenic and adipogenic effect of ghrelin were mainly dependent on the ß-arrestin bound to the phosphorylated GHSR1a. In contrast, the ghrelin function on GH secretion was entirely mediated by G protein signaling. Our data is consistent with the hypothesis that the phosphorylation pattern on the C terminus of GHSR1a determines the signaling and physiological output.2016-04-18T11:32:29ZCPEB and miR-15/16 Co-Regulate Translation of Cyclin E1 mRNA during Xenopus Oocyte MaturationWilczynska, AniaGit, A.Argasinska, J.Belloc, E.Standart, N.http://hdl.handle.net/2381/373242016-04-16T02:23:38Z2016-04-15T16:13:15ZTitle: CPEB and miR-15/16 Co-Regulate Translation of Cyclin E1 mRNA during Xenopus Oocyte Maturation
Authors: Wilczynska, Ania; Git, A.; Argasinska, J.; Belloc, E.; Standart, N.
Abstract: Cell cycle transitions spanning meiotic maturation of the Xenopus oocyte and early embryogenesis are tightly regulated at the level of stored inactive maternal mRNA. We investigated here the translational control of cyclin E1, required for metaphase II arrest of the unfertilised egg and the initiation of S phase in the early embryo. We show that the cyclin E1 mRNA is regulated by both cytoplasmic polyadenylation elements (CPEs) and two miR-15/16 target sites within its 3’UTR. Moreover, we provide evidence that maternal miR-15/16 microRNAs co-immunoprecipitate with CPE-binding protein (CPEB), and that CPEB interacts with the RISC component Ago2. Experiments using competitor RNA and mutated cyclin E1 3’UTRs suggest cooperation of the regulatory elements to sustain repression of the cyclin E1 mRNA during early stages of maturation when CPEB becomes limiting and cytoplasmic polyadenylation of repressed mRNAs begins. Importantly, injection of anti-miR-15/16 LNA results in the early polyadenylation of endogenous cyclin E1 mRNA during meiotic maturation, and an acceleration of GVBD, altogether strongly suggesting that the proximal CPEB and miRNP complexes act to mutually stabilise each other. We conclude that miR-15/16 and CPEB co-regulate cyclin E1 mRNA. This is the first demonstration of the co-operation of these two pathways.2016-04-15T16:13:15ZSuppression of human T cell proliferation by the caspase inhibitors, z-VAD-FMK and z-IETD-FMK is independent of their caspase inhibition propertiesLawrence, C. P.Chow, S. C.http://hdl.handle.net/2381/373172016-04-15T02:01:22Z2016-04-14T13:28:08ZTitle: Suppression of human T cell proliferation by the caspase inhibitors, z-VAD-FMK and z-IETD-FMK is independent of their caspase inhibition properties
Authors: Lawrence, C. P.; Chow, S. C.
Abstract: The caspase inhibitors, benzyloxycarbony (Cbz)-l-Val-Ala-Asp (OMe)-fluoromethylketone (z-VAD-FMK) and benzyloxycarbonyl (Cbz)-Ile-Glu (OMe)-Thr-Asp (OMe)-FMK (z-IETD-FMK) at non-toxic doses were found to be immunosuppressive and inhibit human T cell proliferation induced by mitogens and IL-2 in vitro. Both caspase inhibitors were shown to block NF-κB in activated primary T cells, but have little inhibitory effect on the secretion of IL-2 and IFN-γ during T cell activation. However, the expression of IL-2 receptor α-chain (CD25) in activated T cells was inhibited by both z-VAD-FMK and z-IETD-FMK, whereas the expression of the early activated T cell marker, CD69 was unaffected. During primary T cell activation via the antigen receptor, both caspase-8 and caspase-3 were activated and processed to their respective subunits, but neither caspase inhibitors had any effect on the processing of these two caspases. In sharp contrast both caspase inhibitors readily blocked apoptosis and the activation of caspases during FasL-induced apoptosis in activated primary T cells and Jurkat T cells. Collectively, the results demonstrate that both z-VAD-FMK and z-IETD-FMK are immunosuppressive in vitro and inhibit T cell proliferation without blocking the processing of caspase-8 and caspase-3.2016-04-14T13:28:08ZA systematic approach for testing expression of human full-length proteins in cell-free expression systemsLanglais, ClaudiaGuilleaume, B.Wermke, N.Scheuermann, T.Ebert, L.LaBaer, J.Korn, B.http://hdl.handle.net/2381/372852016-04-14T02:01:00Z2016-04-13T12:10:24ZTitle: A systematic approach for testing expression of human full-length proteins in cell-free expression systems
Authors: Langlais, Claudia; Guilleaume, B.; Wermke, N.; Scheuermann, T.; Ebert, L.; LaBaer, J.; Korn, B.
Abstract: Background
The growing field of proteomics and systems biology is resulting in an ever increasing demand for purified recombinant proteins for structural and functional studies. Here, we show a systematic approach to successfully express a full-length protein of interest by using cell-free and cell-based expression systems.
Results
In a pre-screen, we evaluated the expression of 960 human full-length open reading frames in Escherichia coli (in vivo and in vitro). After analysing the protein expression rate and solubility, we chose a subset of 87 plasmids yielding no protein product in E. coli in vivo. These targets were subjected to a more detailed analysis comparing a prokaryotic cell-free E. coli system with an eukaryotic wheat germ system. In addition, we determined the expression rate, yield and solubility of those proteins. After sequence optimisation for the E. coli in vitro system and generating linear templates for wheat germ expression, the success rate of cell-free protein expression reached 93%.
Conclusion
We have demonstrated that protein expression in cell-free systems is an appropriate technology for the successful expression of soluble full-length proteins. In our study, wheat germ expression using a two compartment system is the method of choice as it shows high solubility and high protein yield.2016-04-13T12:10:24ZTissue-specific expression of p73 C-terminal isoforms in miceGrespi, FrancescaAmelio, IvanoTucci, PaolaAnnicchiarico-Petruzzelli, M.Melino, Gerryhttp://hdl.handle.net/2381/369042016-02-27T04:39:02Z2016-02-26T10:36:02ZTitle: Tissue-specific expression of p73 C-terminal isoforms in mice
Authors: Grespi, Francesca; Amelio, Ivano; Tucci, Paola; Annicchiarico-Petruzzelli, M.; Melino, Gerry
Abstract: p73 is a p53 family transcription factor. Due to the presence in the 5′ flanking region of two promoters, there are two N-terminal variants, TAp73, which retains a fully active transactivation domain (TA), and ΔNp73, in which the N terminus is truncated. In addition, extensive 3′ splicing gives rise to at least seven distinctive isoforms; TAp73-selective knockout highlights its role as a regulator of cell death, senescence and tumor suppressor. ΔNp73-selective knockout, on the other hand, highlights anti-apoptotic function of ΔNp73 and its involvement in DNA damage response. In this work, we investigated the expression pattern of murine p73 C-terminal isoforms. By using a RT-PCR approach, we were able to detect mRNAs of all the C-terminal isoforms described in humans. We characterized their in vivo expression profile in mouse organs and in different mouse developmental stages. Finally, we investigated p73 C-terminal expression profile following DNA damage, ex vivo after primary cultures treatment and in vivo after systemic administration of cytotoxic compounds. Overall, our study first elucidates spatio-temporal expression of mouse p73 isoforms and provides novel insights on their expression-switch under triggered conditions.
Description: Supplemental materials may be found here:
www.landesbioscience.com/admin/article/22787/2016-02-26T10:36:02Zp63 the guardian of human reproductionAmelio, IvanoGrespi, FrancescaAnnicchiarico-Petruzzelli, M.Melino, Gerryhttp://hdl.handle.net/2381/369032016-02-27T04:39:36Z2016-02-26T10:29:17ZTitle: p63 the guardian of human reproduction
Authors: Amelio, Ivano; Grespi, Francesca; Annicchiarico-Petruzzelli, M.; Melino, Gerry
Abstract: p63 is a transcriptional factor implicated in cancer and development. The presence in TP63 gene of alternative promoters allows expression of one isoform containing the N-terminal transactivation domain (TA isoform) and one N-terminal truncated isoform (ΔN isoform). Complete ablation of all p63 isoforms produced mice with fatal developmental abnormalities, including lack of epidermal barrier, limbs and other epidermal appendages. Specific TAp63-null mice, although they developed normally, failed to undergo in DNA damage-induced apoptosis during primordial follicle meiotic arrest, suggesting a p63 involvement in maternal reproduction. Recent findings have elucidated the role in DNA damage response of a novel Hominidae p63 isoform, GTAp63, specifically expressed in human spermatic precursors. Thus, these findings suggest a unique strategy of p63 gene, to evolve in order to preserve the species as a guardian of reproduction. Elucidation of the biological basis of p63 function in reproduction may provide novel approaches to the control of human fertility.2016-02-26T10:29:17ZThe “Sharp” blade against HIF-mediated metastasisAmelio, IvanoMelino, Gerryhttp://hdl.handle.net/2381/369022016-02-27T04:39:03Z2016-02-26T10:18:51ZTitle: The “Sharp” blade against HIF-mediated metastasis
Authors: Amelio, Ivano; Melino, Gerry
Abstract: Hypoxia-inducible factors (HIFs) control cellular adaptation to oxygen deprivation. Cancer cells engage HIFs to sustain their growth in adverse conditions, thus promoting a cellular reprograming that includes metabolism, proliferation, survival and mobility. HIFs overexpression in human cancer biopsies correlates with high metastasis and mortality. A recent report has elucidated a novel mechanism for HIFs regulation in triple-negative breast cancer. Specifically, the basic helix-loop-helix (bHLH), Sharp-1, serves HIF1α to the proteasome and promotes its O2-indendpendet degradation, counteracting HIF-mediated metastasis. These findings shed light on how HIFs are manipulated during cancer pathogenesis.2016-02-26T10:18:51ZSpecificity factors in cytoplasmic polyadenylationCharlesworth, A.Meijer, Hedda A.de Moor, C. H.http://hdl.handle.net/2381/369002016-02-26T03:28:12Z2016-02-25T15:39:15ZTitle: Specificity factors in cytoplasmic polyadenylation
Authors: Charlesworth, A.; Meijer, Hedda A.; de Moor, C. H.
Abstract: Poly(A) tail elongation after export of an messenger RNA (mRNA) to the cytoplasm is called cytoplasmic polyadenylation. It was first discovered in oocytes and embryos, where it has roles in meiosis and development. In recent years, however, has been implicated in many other processes, including synaptic plasticity and mitosis. This review aims to introduce cytoplasmic polyadenylation with an emphasis on the factors and elements mediating this process for different mRNAs and in different animal species. We will discuss the RNA sequence elements mediating cytoplasmic polyadenylation in the 3′ untranslated regions of mRNAs, including the CPE, MBE, TCS, eCPE, and C-CPE. In addition to describing the role of general polyadenylation factors, we discuss the specific RNA binding protein families associated with cytoplasmic polyadenylation elements, including CPEB (CPEB1, CPEB2, CPEB3, and CPEB4), Pumilio (PUM2), Musashi (MSI1, MSI2), zygote arrest (ZAR2), ELAV like proteins (ELAVL1, HuR), poly(C) binding proteins (PCBP2, αCP2, hnRNP-E2), and Bicaudal C (BICC1). Some emerging themes in cytoplasmic polyadenylation will be highlighted. To facilitate understanding for those working in different organisms and fields, particularly those who are analyzing high throughput data, HUGO gene nomenclature for the human orthologs is used throughout. Where human orthologs have not been clearly identified, reference is made to protein families identified in man.2016-02-25T15:39:15ZThe small molecule dispergo tubulates the endoplasmic reticulum and inhibits exportLu, L.Hannoush, R. N.Goess, B. C.Varadarajan, ShankarShair, M. D.Kirchhausen, T.http://hdl.handle.net/2381/368812016-02-26T03:28:13Z2016-02-25T12:30:52ZTitle: The small molecule dispergo tubulates the endoplasmic reticulum and inhibits export
Authors: Lu, L.; Hannoush, R. N.; Goess, B. C.; Varadarajan, Shankar; Shair, M. D.; Kirchhausen, T.
Abstract: The mammalian endoplasmic reticulum (ER) is an organelle that maintains a complex, compartmentalized organization of interconnected cisternae and tubules while supporting a continuous flow of newly synthesized proteins and lipids to the Golgi apparatus. Using a phenotypic screen, we identify a small molecule, dispergo, that induces reversible loss of the ER cisternae and extensive ER tubulation, including formation of ER patches comprising densely packed tubules. Dispergo also prevents export from the ER to the Golgi apparatus, and this traffic block results in breakdown of the Golgi apparatus, primarily due to maintenance of the constitutive retrograde transport of its components to the ER. The effects of dispergo are reversible, since its removal allows recovery of the ER cisternae at the expense of the densely packed tubular ER patches. This recovery occurs together with reactivation of ER-to-Golgi traffic and regeneration of a functional Golgi with correct morphology. Because dispergo is the first small molecule that reversibly tubulates the ER and inhibits its export function, it will be useful in studying these complex processes.
Description: Supplemental Material can be found at: http://www.molbiolcell.org/content/suppl/2013/02/04/mbc.E12-08-0575.DC1.html2016-02-25T12:30:52ZCRISPR: a new method for genetic engineering – A prokaryotic immune component may potentially open a new era of gene silencingAmelio, I.Melino, G.http://hdl.handle.net/2381/368212016-02-24T03:25:07Z2016-02-23T11:54:46ZTitle: CRISPR: a new method for genetic engineering – A prokaryotic immune component may potentially open a new era of gene silencing
Authors: Amelio, I.; Melino, G.2016-02-23T11:54:46ZClinical update on cancer: molecular oncology of head and neck cancerSuh, Y.Amelio, I.Guerrero Urbano, T.Tavassoli, M.http://hdl.handle.net/2381/368192016-02-24T03:25:15Z2016-02-23T11:39:45ZTitle: Clinical update on cancer: molecular oncology of head and neck cancer
Authors: Suh, Y.; Amelio, I.; Guerrero Urbano, T.; Tavassoli, M.
Abstract: Head and neck cancers encompass a heterogeneous group of tumours that, in general, are biologically aggressive in nature. These cancers remain difficult to treat and treatment can cause severe, long-term side effects. For patients who are not cured by surgery and/or (chemo)radiotherapy, there are few effective treatment options. Targeted therapies and predictive biomarkers are urgently needed in order to improve the management and minimise the treatment toxicity, and to allow selection of patients who are likely to benefit from both nonselective and targeted therapies. This clinical update aims to provide an insight into the current understanding of the molecular pathogenesis of the disease, and explores the novel therapies under development and in clinical trials.2016-02-23T11:39:45ZIdentification of LDH-A as a therapeutic target for cancer cell killing via (i) p53/NAD(H)-dependent and (ii) p53-independent pathwaysAllison, S. J.Knight, J. R. P.Granchi, C.Rani, R.Minutolo, F.Milner, J .Phillips, R. M.http://hdl.handle.net/2381/368152016-02-24T03:25:02Z2016-02-23T09:40:59ZTitle: Identification of LDH-A as a therapeutic target for cancer cell killing via (i) p53/NAD(H)-dependent and (ii) p53-independent pathways
Authors: Allison, S. J.; Knight, J. R. P.; Granchi, C.; Rani, R.; Minutolo, F.; Milner, J .; Phillips, R. M.
Abstract: Most cancer cells use aerobic glycolysis to fuel their growth. The enzyme lactate dehydrogenase-A (LDH-A) is key to cancer’s glycolytic phenotype, catalysing the regeneration of nicotinamide adenine dinucleotide (NAD+) from reduced nicotinamide adenine dinucleotide (NADH) necessary to sustain glycolysis. As such, LDH-A is a promising target for anticancer therapy. Here we ask if the tumour suppressor p53, a major regulator of cellular metabolism, influences the response of cancer cells to LDH-A suppression. LDH-A knockdown by RNA interference (RNAi) induced cancer cell death in p53 wild-type, mutant and p53-null human cancer cell lines, indicating that endogenous LDH-A promotes cancer cell survival irrespective of cancer cell p53 status. Unexpectedly, however, we uncovered a novel role for p53 in the regulation of cancer cell NAD+ and its reduced form NADH. Thus, LDH-A silencing by RNAi, or its inhibition using a small-molecule inhibitor, resulted in a p53-dependent increase in the cancer cell ratio of NADH:NAD+. This effect was specific for p53+/+ cancer cells and correlated with (i) reduced activity of NAD+-dependent deacetylase sirtuin 1 (SIRT1) and (ii) an increase in acetylated p53, a known target of SIRT1 deacetylation activity. In addition, activation of the redox-sensitive anticancer drug EO9 was enhanced selectively in p53+/+ cancer cells, attributable to increased activity of NAD(P)H-dependent oxidoreductase NQO1 (NAD(P)H quinone oxidoreductase 1). Suppressing LDH-A increased EO9-induced DNA damage in p53+/+ cancer cells, but importantly had no additive effect in non-cancer cells. Our results identify a unique strategy by which the NADH/NAD+ cellular redox status can be modulated in a cancer-specific, p53-dependent manner and we show that this can impact upon the activity of important NAD(H)-dependent enzymes. To summarise, this work indicates two distinct mechanisms by which suppressing LDH-A could potentially be used to kill cancer cells selectively, (i) through induction of apoptosis, irrespective of cancer cell p53 status and (ii) as a part of a combinatorial approach with redox-sensitive anticancer drugs via a novel p53/NAD(H)-dependent mechanism.
Description: Supplementary Information accompanies this paper on the Oncogenesis website http://www.nature.com/oncsis/journal/v3/n5/suppinfo/oncsis201416s1.html2016-02-23T09:40:59ZPrions: Generation and Spread Versus NeurotoxicityHalliday, MarkRadford, HeloisMallucci, Giovanna R.http://hdl.handle.net/2381/368142016-02-24T03:25:15Z2016-02-23T09:23:50ZTitle: Prions: Generation and Spread Versus Neurotoxicity
Authors: Halliday, Mark; Radford, Helois; Mallucci, Giovanna R.
Abstract: Neurodegenerative diseases are characterized by the aggregation of misfolded proteins in the brain. Among these disorders are the prion diseases, which are transmissible, and in which the misfolded proteins (“prions”) are also the infectious agent. Increasingly, it appears that misfolded proteins in Alzheimer and Parkinson diseases and the tauopathies also propagate in a “prion-like” manner. However, the association between prion formation, spread, and neurotoxicity is not clear. Recently, we showed that in prion disease, protein misfolding leads to neurodegeneration through dysregulation of generic proteostatic mechanisms, specifically, the unfolded protein response. Genetic and pharmacological manipulation of the unfolded protein response was neuroprotective despite continuing prion replication, hence dissociating this from neurotoxicity. The data have clear implications for treatment across the spectrum of these disorders, targeting pathogenic processes downstream of protein misfolding.2016-02-23T09:23:50ZPERK inhibition prevents tau-mediated neurodegeneration in a mouse model of frontotemporal dementiaRadford, HeloisMoreno, Julie A.Verity, NicholasHalliday, MarkMallucci, Giovanna R.http://hdl.handle.net/2381/366862016-02-11T03:34:22Z2016-02-10T15:11:00ZTitle: PERK inhibition prevents tau-mediated neurodegeneration in a mouse model of frontotemporal dementia
Authors: Radford, Helois; Moreno, Julie A.; Verity, Nicholas; Halliday, Mark; Mallucci, Giovanna R.
Abstract: The PERK-eIF2α branch of the Unfolded Protein Response (UPR) mediates the transient shutdown of translation in response to rising levels of misfolded proteins in the endoplasmic reticulum. PERK and eIF2α activation are increasingly recognised in postmortem analyses of patients with neurodegenerative disorders, including Alzheimer’s disease, the tauopathies and prion disorders. These are all characterised by the accumulation of misfolded disease-specific proteins in the brain in association with specific patterns of neuronal loss, but the role of UPR activation in their pathogenesis is unclear. In prion-diseased mice, overactivation of PERK-P/eIF2α-P signalling results in the sustained reduction in global protein synthesis, leading to synaptic failure, neuronal loss and clinical disease. Critically, restoring vital neuronal protein synthesis rates by inhibiting the PERK-eIF2α pathway, both genetically and pharmacologically, prevents prion neurodegeneration downstream of misfolded prion protein accumulation. Here we show that PERK-eIF2α-mediated translational failure is a key process leading to neuronal loss in a mouse model of frontotemporal dementia, where the misfolded protein is a form of mutant tau. rTg4510 mice, which overexpress the P301L tau mutation, show dysregulated PERK signalling and sustained repression of protein synthesis by 6 months of age, associated with onset of neurodegeneration. Treatment with the PERK inhibitor, GSK2606414, from this time point in mutant tau-expressing mice restores protein synthesis rates, protecting against further neuronal loss, reducing brain atrophy and abrogating the appearance of clinical signs. Further, we show that PERK-eIF2α activation also contributes to the pathological phosphorylation of tau in rTg4510 mice, and that levels of phospho-tau are lowered by PERK inhibitor treatment, providing a second mechanism of protection. The data support UPR-mediated translational failure as a generic pathogenic mechanism in protein-misfolding disorders, including tauopathies, that can be successfully targeted for prevention of neurodegeneration.2016-02-10T15:11:00ZThe in vitro respiratory toxicity of cristobalite-bearing volcanic ashDamby, D. E.Murphy, Fiona A.Horwell, C. J.Raftis, J.Donaldson, K.http://hdl.handle.net/2381/366832016-02-11T03:34:11Z2016-02-10T14:56:19ZTitle: The in vitro respiratory toxicity of cristobalite-bearing volcanic ash
Authors: Damby, D. E.; Murphy, Fiona A.; Horwell, C. J.; Raftis, J.; Donaldson, K.
Abstract: Ash from dome-forming volcanoes poses a unique hazard to millions of people worldwide due to an abundance of respirable cristobalite, a crystalline silica polymorph. Crystalline silica is an established respiratory hazard in other mixed dusts, but its toxicity strongly depends on sample provenance. Previous studies suggest that cristobalite-bearing volcanic ash is not as bio-reactive as may be expected for a dust containing crystalline silica. We systematically address the hazard posed by volcanic cristobalite by analysing a range of dome-related ash samples, and interpret the crystalline silica hazard according to the mineralogical nature of volcanic cristobalite. Samples are sourced from five well-characterized dome-forming volcanoes that span a range of magmatic compositions, specifically selecting samples rich in cristobalite (up to 16 wt%). Isolated respirable fractions are used to investigate the in vitro response of THP-1 macrophages and A549 type II epithelial cells in cytotoxicity, cellular stress, and pro-inflammatory assays associated with crystalline silica toxicity. Dome-related ash is minimally reactive in vitro for a range of source compositions and cristobalite contents. Cristobalite-based toxicity is not evident in the assays employed, supporting the notion that crystalline silica provenance influences reactivity. Macrophages experienced minimal ash-induced cytotoxicity and intracellular reduction of glutathione; however, production of IL-1β, IL-6 and IL-8 were sample-dependent. Lung epithelial cells experienced moderate apoptosis, sample-dependent reduction of glutathione, and minimal cytokine production. We suggest that protracted interaction between particles and epithelial cells may never arise due to effective clearance by macrophages. However, volcanic ash has the propensity to incite a low, but significant, and sample-dependent response; the effect of this response in vivo is unknown and prolonged exposure may yet pose a hazard.2016-02-10T14:56:19ZMicroRNAs and p63 in epithelial stemnessCandi, E.Amelio, I.Agostini, M.Melino, G.http://hdl.handle.net/2381/366792016-02-11T03:34:09Z2016-02-10T14:34:41ZTitle: MicroRNAs and p63 in epithelial stemness
Authors: Candi, E.; Amelio, I.; Agostini, M.; Melino, G.
Abstract: MicroRNAs (miRs) are a class of small noncoding RNAs that suppress the expression of protein-coding genes by repressing protein translation. Although the roles that miRs and the miR processing machinery have in regulating epithelial stem cell biology are not fully understood, their fundamental contributions to these processes have been demonstrated over the last few years. The p53-family member p63 is an essential transcription factor for epidermal morphogenesis and homeostasis. p63 functions as a determinant for keratinocyte cell fate and helps to regulate the balance between stemness, differentiation and senescence. An important factor that regulates p63 function is the reciprocal interaction between p63 and miRs. Some miRs control p63 expression, and p63 regulates the miR expression profile in the epidermis. p63 controls miR expression at different levels. It directly regulates the transcription of several miRs and indirectly regulates their processing by regulating the expression of the miR processing components Dicer and DGCR8. In this review, we will discuss the recent findings on the miR–p63 interaction in epidermal biology, particularly focusing on the ΔNp63-dependent regulation of DGCR8 recently described in the ΔNp63−/− mouse. We provide a unified view of the current knowledge and discuss the apparent discrepancies and perspective therapeutic opportunities.2016-02-10T14:34:41ZReview: Modulating the unfolded protein response to prevent neurodegeneration and enhance memoryHalliday, MarkMallucci, Giovanna R.http://hdl.handle.net/2381/366542016-02-11T03:34:10Z2016-02-10T10:47:34ZTitle: Review: Modulating the unfolded protein response to prevent neurodegeneration and enhance memory
Authors: Halliday, Mark; Mallucci, Giovanna R.
Abstract: Recent evidence has placed the unfolded protein response (UPR) at the centre of pathological processes leading to neurodegenerative disease. The translational repression caused by UPR activation starves neurons of the essential proteins they need to function and survive. Restoration of protein synthesis, via genetic or pharmacological means, is neuroprotective in animal models, prolonging survival. This is of great interest due to the observation of UPR activation in the post mortem brains of patients with Alzheimer's, Parkinson's, tauopathies and prion diseases. Protein synthesis is also an essential step in the formation of new memories. Restoring translation in disease or increasing protein synthesis from basal levels has been shown to improve memory in numerous models. As neurodegenerative diseases often present with memory impairments, targeting the UPR to both provide neuroprotection and enhance memory provides an extremely exciting novel therapeutic target.2016-02-10T10:47:34ZLow-Level Lead Exposure and Intellectual Impairment in Children: Koller et al. RespondKoller, KarinLevy, LenBrown, TerrySpurgeon, A.http://hdl.handle.net/2381/365042016-02-02T03:30:48Z2016-02-01T16:57:15ZTitle: Low-Level Lead Exposure and Intellectual Impairment in Children: Koller et al. Respond
Authors: Koller, Karin; Levy, Len; Brown, Terry; Spurgeon, A.2016-02-01T16:57:15ZThe Aryl Hydrocarbon Receptor: Differential Contribution to T Helper 17 and T Cytotoxic 17 Cell DevelopmentHayes, Mark D.Ovcinnikovs, VitalijsSmith, Andrew G.Kimber, IanDearman, Rebecca J.http://hdl.handle.net/2381/361802016-01-07T03:09:16Z2016-01-06T10:53:54ZTitle: The Aryl Hydrocarbon Receptor: Differential Contribution to T Helper 17 and T Cytotoxic 17 Cell Development
Authors: Hayes, Mark D.; Ovcinnikovs, Vitalijs; Smith, Andrew G.; Kimber, Ian; Dearman, Rebecca J.
Abstract: The aryl hydrocarbon receptor (AhR) has been shown to be required for optimal Thelper (Th) 17 cell activation. Th17 cells provide immunity against extracellular pathogens and are implicated in autoimmune diseases. Herein, the role of the AhR in cytokine production by Th17, and by the analogous population of T cytotoxic (Tc)17 cells, has been examined. Lymph node Tc (CD8+) and Th (CD4+) cells were isolated by negative selection from naive AhR+/− and AhR−/− mice and polarised to Tc1/Th1 or Tc17/Th17 phenotypes with appropriate cytokines. Cell differentiation was assessed as a function of mRNA and protein (ELISA and flow cytometry) expression for interferon (IFN)-γ and for key Th17 cytokines. In AhR+/− mice, Th17 cells displayed an exclusive IL-17 profile, which was markedly inhibited by a selective AhR antagonist to levels observed in AhR knockout mice. Addition of the natural AhR agonist 6-formylindolo[3,2-b]carbazole (FICZ) markedly enhanced Th17 cell activity in the heterozygotes. In contrast, Tc17 cells polarised into 3 distinct subsets: producing either IL-17 or IFN-γ alone, or both cytokines. Blocking AhR was also detrimental to Tc17 development, with reduced responses recorded in AhR−/− mice and antagonist-mediated reduction of IL-17 expression in the heterozygotes. However, Tc17 cells were largely refractory to exogenous FICZ, presumably because Tc17 cells express baseline AhR mRNA, but unlike Th17 cells, there is no marked up-regulation during polarisation. Thus, Th17 cell development is more dependent upon AhR activation than is Tc17 cell development, suggesting that endogenous AhR ligands play a much greater role in driving Th17 cell responses.2016-01-06T10:53:54ZGlycogen Synthase Kinase-3 Inhibition Enhances Translation of Pluripotency-Associated Transcription Factors to Contribute to Maintenance of Mouse Embryonic Stem Cell Self-RenewalSanchez-Ripoll, YolandaBone, Heather K.Owen, TomGuedes, Ana M. V.Abranches, ElsaKumpfmueller, BenjaminSpriggs, Ruth V.Henrique, DomingosWelham, Melanie J.http://hdl.handle.net/2381/361792016-01-07T03:09:06Z2016-01-06T10:49:11ZTitle: Glycogen Synthase Kinase-3 Inhibition Enhances Translation of Pluripotency-Associated Transcription Factors to Contribute to Maintenance of Mouse Embryonic Stem Cell Self-Renewal
Authors: Sanchez-Ripoll, Yolanda; Bone, Heather K.; Owen, Tom; Guedes, Ana M. V.; Abranches, Elsa; Kumpfmueller, Benjamin; Spriggs, Ruth V.; Henrique, Domingos; Welham, Melanie J.
Abstract: Maintenance of embryonic stem cell (ESC) self-renewal and pluripotency are controlled by extrinsic factors, molecular signaling pathways and transcriptional regulators. While many of the key players have been studied in depth, how the molecular signals interact with transcription factors of the pluripotency network to regulate their action remains less well understood. Inhibition of glycogen synthase kinase 3 (Gsk-3) has been implicated in the maintenance of mouse ESC pluripotency, although there is contradictory data on its role, with enhancement of cell survival and metabolism, stabilisation of c-Myc and activation of Wnt signalling proposed as potential mechanisms. We have discovered that suppression of Gsk-3 activity leads to enhanced protein levels of key transcriptional regulators of the pluripotency network, notably Nanog, Tbx3 and c-Myc. Protein stability was unchanged following Gsk-3 inhibition, although interestingly, Nanog and Tbx3 proteins were found to have half-lives of 1–3 h, while that of Oct4 protein was longer, at 6 h. We demonstrate that the effects on protein levels seen following inhibition of Gsk-3 are due to both enhanced de novo synthesis of Nanog protein and increases in the proportion of Nanog and Tbx3 RNAs bound to polysomes, findings consistent with Gsk-3 regulating translation of these factors. These effects were not due to changes in regulators of general translation initiation machinery nor mediated via the 5′ or 3′ UTR sequences of Nanog alone. The data we present provide both new conceptual insight into the mechanisms regulated by Gsk-3 that may contribute to ESC self-renewal and, importantly, establish control of protein translation as an additional mechanism involved in modulation of ESC pluripotency.2016-01-06T10:49:11ZmiR-24 affects hair follicle morphogenesis targeting Tcf-3Amelio, I.Lena, A. M.Bonanno, E.Melino, G.Candi, E.http://hdl.handle.net/2381/361772016-01-07T03:09:25Z2016-01-06T10:42:17ZTitle: miR-24 affects hair follicle morphogenesis targeting Tcf-3
Authors: Amelio, I.; Lena, A. M.; Bonanno, E.; Melino, G.; Candi, E.
Abstract: During embryonic development, hair follicles (HFs) develop from an epidermal–mesenchymal cross talk between the ectoderm progenitor layer and the underlying dermis. Epidermal stem cell activation represents a crucial point both for HF morphogenesis and for hair regeneration. miR-24 is an anti-proliferative microRNA (miRNA), which is induced during differentiation of several cellular systems including the epidermis. Here, we show that miR-24 is expressed in the HF and has a role in hair morphogenesis. We generated transgenic mice ectopically expressing miR-24 under the K5 promoter. The K5::miR-24 animals display a marked defect in HF morphogenesis, with thinning of hair coat and altered HF structure. Expression of miR-24 alters the normal process of hair keratinocyte differentiation, leading to altered expression of differentiation markers. MiR-24 directly represses the hair keratinocyte stemness regulator Tcf-3. These results support the notion that microRNAs, and among them miR-24, have an important role in postnatal epidermal homeostasis.2016-01-06T10:42:17ZA novel extracellular role for tissue transglutaminase in matrix-bound VEGF-mediated angiogenesisWang, Z.Perez, M.Caja, S.Melino, G.Johnson, T. S.Lindfors, K.Griffin, M.http://hdl.handle.net/2381/361752016-01-07T03:09:05Z2016-01-06T10:35:20ZTitle: A novel extracellular role for tissue transglutaminase in matrix-bound VEGF-mediated angiogenesis
Authors: Wang, Z.; Perez, M.; Caja, S.; Melino, G.; Johnson, T. S.; Lindfors, K.; Griffin, M.
Abstract: The importance of tissue transglutaminase (TG2) in angiogenesis is unclear and contradictory. Here we show that inhibition of extracellular TG2 protein crosslinking or downregulation of TG2 expression leads to inhibition of angiogenesis in cell culture, the aorta ring assay and in vivo models. In a human umbilical vein endothelial cell (HUVEC) co-culture model, inhibition of extracellular TG2 activity can halt the progression of angiogenesis, even when introduced after tubule formation has commenced and after addition of excess vascular endothelial growth factor (VEGF). In both cases, this leads to a significant reduction in tubule branching. Knockdown of TG2 by short hairpin (shRNA) results in inhibition of HUVEC migration and tubule formation, which can be restored by add back of wt TG2, but not by the transamidation-defective but GTP-binding mutant W241A. TG2 inhibition results in inhibition of fibronectin deposition in HUVEC monocultures with a parallel reduction in matrix-bound VEGFA, leading to a reduction in phosphorylated VEGF receptor 2 (VEGFR2) at Tyr1214 and its downstream effectors Akt and ERK1/2, and importantly its association with β1 integrin. We propose a mechanism for the involvement of matrix-bound VEGFA in angiogenesis that is dependent on extracellular TG2-related activity.
Description: Supplementary Information accompanies this paper on Cell Death and Disease website http://www.nature.com/cddis/journal/v4/n9/suppinfo/cddis2013318s1.html2016-01-06T10:35:20ZCaspase-1 is a novel target of p63 in tumor suppressionCelardo, I.Grespi, F.Antonov, A.Bernassola, F.Garabadgiu, A. V.Melino, G.Amelio, I.http://hdl.handle.net/2381/361732016-01-07T03:09:17Z2016-01-06T10:14:45ZTitle: Caspase-1 is a novel target of p63 in tumor suppression
Authors: Celardo, I.; Grespi, F.; Antonov, A.; Bernassola, F.; Garabadgiu, A. V.; Melino, G.; Amelio, I.
Abstract: p63 is a p53 family transcription factor, which besides unique roles in epithelial development, shares tumor suppressive activity with its homolog p53. The p63 gene has different transcriptional start sites, which generate two N-terminal isoforms (transactivation domain (TA)p63 and amino terminal truncated protein(ΔN)p63); in addition alternative splicing at the 5′-end give rise to at least five C-terminal isoforms. This complexity of gene structure has probably fostered the debate and controversy on p63 function in cancer, with TP63-harboring two distinctive promoters, codifying for the TAp63 and ΔNp63 isoforms, and having discrete functions. However, ΔNp63 also drives expression of target genes that have a relevant role in cancer and metastasis. In this study, we identified a novel p63 transcriptional target, caspase-1. Caspase-1 is proinflammatory caspase, which functions in tumor suppression. We show that both p63 isoforms promote caspase-1 expression by physical binding to its promoter. Consistent with our in vitro findings, we also identified a direct correlation between p63 and caspase-1 expression in human cancer data sets. In addition, survival estimation analysis demonstrated that functional interaction between p63 and caspase-1 represents a predictor of positive survival outcome in human cancers. Overall, our data report a novel p63 target gene involved in tumor suppression, and the clinical analysis underlines the biological relevance of this finding and suggests a further clinically predictive biomarker.2016-01-06T10:14:45ZCaspases: the executioners of apoptosisCohen, Gerald M.http://hdl.handle.net/2381/359752015-12-02T10:07:54Z2015-12-02T10:07:18ZTitle: Caspases: the executioners of apoptosis
Authors: Cohen, Gerald M.
Abstract: Apoptosis is a major form of cell death, characterized initially by a series of stereotypic morphological changes. In the nematode Caenorhabditis elegans, the gene ced-3 encodes a protein required for developmental cell death. Since the recognition that CED-3 has sequence identity with the mammalian cysteine protease interleukin-1 beta-converting enzyme (ICE), a family of at least 10 related cysteine proteases has been identified. These proteins are characterized by almost absolute specificity for aspartic acid in the P1 position. All the caspases (ICE-like proteases) contain a conserved QACXG (where X is R, Q or G) pentapeptide active-site motif. Capases are synthesized as inactive proenzymes comprising an N-terminal peptide (prodomain) together with one large and one small subunit. The crystal structures of both caspase-1 and caspase-3 show that the active enzyme is a heterotetramer, containing two small and two large subunits. Activation of caspases during apoptosis results in the cleavage of critical cellular substrates, including poly(ADP-ribose) polymerase and lamins, so precipitating the dramatic morphological changes of apoptosis. Apoptosis induced by CD95 (Fas/APO-1) and tumour necrosis factor activates caspase-8 (MACH/FLICE/Mch5), which contains an N-terminus with FADD (Fas-associating protein with death domain)-like death effector domains, so providing a direct link between cell death receptors and the caspases. The importance of caspase prodomains in the regulation of apoptosis is further highlighted by the recognition of adapter molecules, such as RAIDD [receptor-interacting protein (RIP)-associated ICH-1/CED-3-homologous protein with a death domain]/CRADD (caspase and RIP adapter with death domain), which binds to the prodomain of caspase-2 and recruits it to the signalling complex. Cells undergoing apoptosis following triggering of death receptors execute the death programme by activating a hierarchy of caspases, with caspase-8 and possibly caspase-10 being at or near the apex of this apoptotic cascade.2015-12-02T10:07:18ZPrion protein facilitates synaptic vesicle release by enhancing release probabilityRobinson, Susan W.Nugent, Marie L.Dinsdale, DavidSteinert, Joern R.http://hdl.handle.net/2381/359532015-11-26T03:03:05Z2015-11-25T17:09:35ZTitle: Prion protein facilitates synaptic vesicle release by enhancing release probability
Authors: Robinson, Susan W.; Nugent, Marie L.; Dinsdale, David; Steinert, Joern R.
Abstract: The cellular prion protein (PrPC) has been implicated in several neurodegenerative diseases as a result of protein misfolding. In humans, prion disease occurs typically with a sporadic origin where uncharacterized mechanisms induce spontaneous PrPC misfolding leading to neurotoxic PrP-scrapie formation (PrPSC). The consequences of misfolded PrPC signalling are well characterized but little is known about the physiological roles of PrPC and its involvement in disease. Here we investigated wild-type PrPC signalling in synaptic function as well as the effects of a disease-relevant mutation within PrPC (proline-to-leucine mutation at codon 101). Expression of wild-type PrPC at the Drosophila neuromuscular junction leads to enhanced synaptic responses as detected in larger miniature synaptic currents which are caused by enlarged presynaptic vesicles. The expression of the mutated PrPC leads to reduction of both parameters compared with wild-type PrPC. Wild-type PrPC enhances synaptic release probability and quantal content but reduces the size of the ready-releasable vesicle pool. Partially, these changes are not detectable following expression of the mutant PrPC. A behavioural test revealed that expression of either protein caused an increase in locomotor activities consistent with enhanced synaptic release and stronger muscle contractions. Both proteins were sensitive to proteinase digestion. These data uncover new functions of wild-type PrPC at the synapse with a disease-relevant mutation in PrPC leading to diminished functional phenotypes. Thus, our data present essential new information possibly related to prion pathogenesis in which a functional synaptic role of PrPC is compromised due to its advanced conversion into PrPSC thereby creating a lack-of-function scenario.2015-11-25T17:09:35ZCharacterisation and comparison of temporal release profiles of nitric oxide generating donorsBradley, Sophie A.Steinert, Joern R.http://hdl.handle.net/2381/332332015-10-13T02:00:48Z2015-10-12T10:54:33ZTitle: Characterisation and comparison of temporal release profiles of nitric oxide generating donors
Authors: Bradley, Sophie A.; Steinert, Joern R.
Abstract: Background
Nitric oxide (NO) is a vital signalling molecule in a variety of tissues including the neuronal, vascular and reproductive system. However, its high diffusibility and inactivation make characterisation of nitrergic signalling difficult. The use of NO donors is essential to characterise downstream signalling pathways but knowledge of donor release capacities is lacking, thus making comparisons of donor responses difficult.
New method
This study characterises NO profiles of commonly used NO donors. Donors were stored under defined conditions and temporal release profiles detected to allow determination of released NO concentrations.
Results
Using NO-sensitive microsensors we assessed release profiles of NO donors following different storage times and conditions. We found that donors such as NOC-5 and PAPA-NONOate decayed substantially within days, whereas SNP and GSNO showed greater stability releasing consistent levels of NO over days. In all donors tested, the amount of released NO differs between frozen and unfrozen stocks.
Comparison with existing method(s)
Fluorescent and amperometric approaches to measure NO concentrations yield a wide range of levels. However, due to a lack of characterisation of the release profiles, inconsistent effects on NO signalling have been widely documented. Our systematic assessment of release profiles of a range of NO donors therefore provides new essential data allowing for improved and defined investigations of nitrergic signalling.
Conclusions
This is the first systematic comparison of temporal release profiles of different NO donors allowing researchers to compare conditions across different studies and the use of defined NO levels by choosing specific donors and concentrations.2015-10-12T10:54:33ZSerine and glycine metabolism in cancerAmelio, IvanoCutruzzolá, F.Antonov, AlexeyAgostini, MassimilianoMelino, Gerryhttp://hdl.handle.net/2381/332012015-10-09T02:01:11Z2015-10-08T11:18:03ZTitle: Serine and glycine metabolism in cancer
Authors: Amelio, Ivano; Cutruzzolá, F.; Antonov, Alexey; Agostini, Massimiliano; Melino, Gerry
Abstract: Serine and glycine are biosynthetically linked, and together provide the essential precursors for the synthesis of proteins, nucleic acids, and lipids that are crucial to cancer cell growth. Moreover, serine/glycine biosynthesis also affects cellular antioxidative capacity, thus supporting tumour homeostasis. A crucial contribution of serine/glycine to cellular metabolism is through the glycine cleavage system, which refuels one-carbon metabolism; a complex cyclic metabolic network based on chemical reactions of folate compounds. The importance of serine/glycine metabolism is further highlighted by genetic and functional evidence indicating that hyperactivation of the serine/glycine biosynthetic pathway drives oncogenesis. Recent developments in our understanding of these pathways provide novel translational opportunities for drug development, dietary intervention, and biomarker identification of human cancers.2015-10-08T11:18:03ZA caspase-3 'death-switch' in colorectal cancer cells for induced and synchronous tumor apoptosis in vitro and in vivo facilitates the development of minimally invasive cell death biomarkersSimpson, K. L.Cawthorne, C.Zhou, C.Hodgkinson, C. L.Walker, M. J.Trapani, F.Kadirvel, M.Brown, G.Dawson, M. J.MacFarlane, MarionWilliams, K. J.Whetton, A. D.Dive, C.http://hdl.handle.net/2381/328252015-07-30T02:00:33Z2015-07-29T09:13:26ZTitle: A caspase-3 'death-switch' in colorectal cancer cells for induced and synchronous tumor apoptosis in vitro and in vivo facilitates the development of minimally invasive cell death biomarkers
Authors: Simpson, K. L.; Cawthorne, C.; Zhou, C.; Hodgkinson, C. L.; Walker, M. J.; Trapani, F.; Kadirvel, M.; Brown, G.; Dawson, M. J.; MacFarlane, Marion; Williams, K. J.; Whetton, A. D.; Dive, C.
Abstract: Novel anticancer drugs targeting key apoptosis regulators have been developed and are undergoing clinical trials. Pharmacodynamic biomarkers to define the optimum dose of drug that provokes tumor apoptosis are in demand; acquisition of longitudinal tumor biopsies is a significant challenge and minimally invasive biomarkers are required. Considering this, we have developed and validated a preclinical 'death-switch' model for the discovery of secreted biomarkers of tumour apoptosis using in vitro proteomics and in vivo evaluation of the novel imaging probe [[superscript: 18]F]ML-10 for non-invasive detection of apoptosis using positron emission tomography (PET). The 'death-switch' is a constitutively active mutant caspase-3 that is robustly induced by doxycycline to drive synchronous apoptosis in human colorectal cancer cells in vitro or grown as tumor xenografts. Death-switch induction caused caspase-dependent apoptosis between 3 and 24 hours in vitro and regression of 'death-switched' xenografts occurred within 24 h correlating with the percentage of apoptotic cells in tumor and levels of an established cell death biomarker (cleaved cytokeratin-18) in the blood. We sought to define secreted biomarkers of tumor apoptosis from cultured cells using Discovery Isobaric Tag proteomics, which may provide candidates to validate in blood. Early after caspase-3 activation, levels of normally secreted proteins were decreased (e.g. Gelsolin and Midkine) and proteins including CD44 and High Mobility Group protein B1 (HMGB1) that were released into cell culture media in vitro were also identified in the bloodstream of mice bearing death-switched tumors. We also exemplify the utility of the death-switch model for the validation of apoptotic imaging probes using [[superscript: 18]F]ML-10, a PET tracer currently in clinical trials. Results showed increased tracer uptake of [[superscript: 18]F]ML-10 in tumours undergoing apoptosis, compared with matched tumour controls imaged in the same animal. Overall, the death-switch model represents a robust and versatile tool for the discovery and validation of apoptosis biomarkers.2015-07-29T09:13:26ZMonitoring human exposure to 2-hydroxyethylating carcinogensFarmer, Peter B.Cordero, RosaAutrup, H.http://hdl.handle.net/2381/318562016-01-15T13:01:28Z2015-03-11T12:35:15ZTitle: Monitoring human exposure to 2-hydroxyethylating carcinogens
Authors: Farmer, Peter B.; Cordero, Rosa; Autrup, H.
Abstract: It is known that human hemoglobin contains low levels of N-terminal N-(2-hydroxyethyl)valine. Possible sources of this modified amino acid are exposure to ethylene oxide or other 2-hydroxyethylating agents. Although such processes are likely to occur endogenously, the exogenous contribution to the adduct formation is unclear. In order to explore the latter, we have analyzed N-(2-hydroxyethyl)valine in the globin of 49 pregnant women and evaluated the effect of smoking status, area of residence, and glutathione S-transferase M1 genotype on adduct levels. Transplacental transfer of hydroxyethylating agents was also studied by the analysis of umbilical cord hemoglobin. The adduct levels in smokers were significantly higher than those in nonsmokers. The adduct levels in umbilical cord blood globin were quantitatively related to those in maternal blood (maternal:fetal ratio 2.7 in smokers and 2.8 in nonsmokers). In the nonsmokers, there was no statistically significant difference in the adduct level between the urban and rural areas, but the level in suburbia tended to be lower than that in the rural area. In the combined smoker and nonsmoker groups, there was no effect of the glutathione S-transferase M1 genotype on levels of N-(2-hydroxyethyl)valine.
Description: PMCID: PMC14696542015-03-11T12:35:15ZUse of a Multistrain Assay Could Improve the NTP Carcinogenesis BioassayFesting, Michael F. W.http://hdl.handle.net/2381/318542016-01-15T12:59:47Z2015-03-11T12:09:11ZTitle: Use of a Multistrain Assay Could Improve the NTP Carcinogenesis Bioassay
Authors: Festing, Michael F. W.
Abstract: There are often large strain differences in the response of laboratory animals to toxic chemicals and carcinogens, with some strains being totally resistant to dose levels that cause acute toxicity and/or cancer in other strains. The current National Toxicology Program carcinogenesis bioassay (NTP-CB) uses only a single isogenic strain of mice and rats and may therefore miss some carcinogens. New short-term tests to predict mutagenesis and possible carcinogenesis are validated using data from the NTP-CB. If the animal data are inaccurate, it may hinder this validation. The accuracy of the NTP-CB could be improved by using two or more strains of each species without increasing the total number of animals. It would be possible to continue to use sample sizes of 48-50 animals, but subdivide these into groups of 12 animals of 4 different strains (48 animals total) per dose/sex group, for example, instead of 48 identical animals. This would quadruple the number of genotypes without any substantial increase in cost. Such a multistrain "factorial" design would, on average, be statistically more powerful then the present design and should increase the chance of detecting carcinogens that currently may give equivocal results or go undetected because the test animal strains happen to be specifically resistant. When strains differ in response, studies of differences in metabolism, pharmacokinetics, DNA damage/repair, cellular responses, and in some cases identification of genetic loci governing sensitivity may provide biological information on toxic mechanisms that would help in assessing human risk and setting permissible exposure limits. The NTP may have made the world a safer place for F344 rats and B6C3F1 mice. The challenge now is to show that the results can be generalized to other genotypes and that the database can be used to validate short-term tests. A first step could be to explore the importance of strain differences in the carcinogenesis bioassay.
Description: PMCID: PMC15190522015-03-11T12:09:11ZTIS11 family proteins and their roles in posttranscriptional gene regulationBaou, MariaJewell, AndrewMurphy, John J.http://hdl.handle.net/2381/284802013-12-05T02:02:58Z2013-12-04T16:41:32ZTitle: TIS11 family proteins and their roles in posttranscriptional gene regulation
Authors: Baou, Maria; Jewell, Andrew; Murphy, John J.
Abstract: Posttranscriptional regulation of gene expression of mRNAs containing adenine-uridine rich elements (AREs) in their 3' untranslated regions is mediated by a number of different proteins that interact with these elements to either stabilise or destabilise them. The present review concerns the TPA-inducible sequence 11 (TIS11) protein family, a small family of proteins, that appears to interact with ARE-containing mRNAs and promote their degradation. This family of proteins has been extensively studied in the past decade. Studies have focussed on determining their biochemical functions, identifying their target mRNAs, and determining their roles in cell functions and diseases.2013-12-04T16:41:32ZLow-threshold potassium currents stabilize IID-sensitivity in the inferior colliculusKarcz, A.Rübsamen, R.Kopp-Scheinpflug, Corneliahttp://hdl.handle.net/2381/284582013-11-28T02:02:04Z2013-11-27T11:20:34ZTitle: Low-threshold potassium currents stabilize IID-sensitivity in the inferior colliculus
Authors: Karcz, A.; Rübsamen, R.; Kopp-Scheinpflug, Cornelia
Abstract: The inferior colliculus (IC) is a midbrain nucleus that exhibits sensitivity to differences in interaural time and intensity (ITDs and IIDs) and integrates information from the auditory brainstem to provide an unambiguous representation of sound location across the azimuth. Further upstream, in the lateral superior olive (LSO), absence of low-threshold potassium currents in Kcna1[superscript −/−] mice interfered with response onset timing and restricted IID-sensitivity to the hemifield of the excitatory ear. Assuming the IID-sensitivity in the IC to be at least partly inherited from LSO neurons, the IC IID-encoding was compared between wild-type (Kcna1[superscript +/+]) and Kcna1[superscript −/−] mice. We asked whether the effect observed in the Kcna1[superscript −/−] LSO is (1) simply propagated into the IC, (2) is enhanced and amplified or, (3) alternatively, is compensated and so no longer detectable. Our results show that general IC response properties as well as the distribution of IID-functions were comparable in Kcna1[superscript −/−] and Kcna1[superscript+/+] mice. In agreement with the literature IC neurons exhibited a higher level-invariance of IID-sensitivity compared to LSO neurons. However, manipulating the timing between the inputs of the two ears caused significantly larger shifts of IID-sensitivity in Kcna1[superscript −/−] mice, whereas in the wild-type IC the IID functions were stable and less sensitive to changes of the temporal relationship between the binaural inputs. We conclude that the IC not only inherits IID-sensitivity from the LSO, but that the convergence with other, non-olivary inputs in the wild-type IC acts to quality-control, consolidate, and stabilize IID representation; this necessary integration of inputs is impaired in the absence of the low-threshold potassium currents mediated by Kv1.1.2013-11-27T11:20:34ZMetabolic effects of TiO₂ nanoparticles, a common component of sunscreens and cosmetics, on human keratinocytesTucci, P.Porta, G.Agostini, M.Dinsdale, D.Iavicoli, I.Cain, K.Finazzi-Agro, A.Melino, G.Willis, A.http://hdl.handle.net/2381/284252015-07-08T10:34:31Z2013-11-19T14:45:43ZTitle: Metabolic effects of TiO₂ nanoparticles, a common component of sunscreens and cosmetics, on human keratinocytes
Authors: Tucci, P.; Porta, G.; Agostini, M.; Dinsdale, D.; Iavicoli, I.; Cain, K.; Finazzi-Agro, A.; Melino, G.; Willis, A.
Abstract: The long-term health risks of nanoparticles remain poorly understood, which is a serious concern given their prevalence in the environment from increased industrial and domestic use. The extent to which such compounds contribute to cellular toxicity is unclear, and although it is known that induction of oxidative stress pathways is associated with this process, the proteins and the metabolic pathways involved with nanoparticle-mediated oxidative stress and toxicity are largely unknown. To investigate this problem further, the effect of TiO[subscript 2] on the HaCaT human keratinocyte cell line was examined. The data show that although TiO[subscript 2] does not affect cell cycle phase distribution, nor cell death, these nanoparticles have a considerable and rapid effect on mitochondrial function. Metabolic analysis was performed to identify 268 metabolites of the specific pathways involved and 85 biochemical metabolites were found to be significantly altered, many of which are known to be associated with the cellular stress response. Importantly, the uptake of nanoparticles into the cultured cells was restricted to phagosomes, TiO[subscript 2] nanoparticles did not enter into the nucleus or any other cytoplasmic organelle. No other morphological changes were detected after 24-h exposure consistent with a specific role of mitochondria in this response.2013-11-19T14:45:43ZTumor suppressor WWOX binds to ΔNp63α and sensitizes cancer cells to chemotherapySalah, Z.Bar-mag, T.Kohn, Y.Pichiorri, F.Palumbo, T.Melino, GerryAqeilan, R.A.http://hdl.handle.net/2381/284242013-11-20T02:05:08Z2013-11-19T14:30:30ZTitle: Tumor suppressor WWOX binds to ΔNp63α and sensitizes cancer cells to chemotherapy
Authors: Salah, Z.; Bar-mag, T.; Kohn, Y.; Pichiorri, F.; Palumbo, T.; Melino, Gerry; Aqeilan, R.A.
Abstract: The WWOX tumor suppressor is a WW domain-containing protein. Its function in the cell has been shown to be mediated, in part, by interacting with its partners through its first WW (WW1) domain. Here, we demonstrated that WWOX via WW1 domain interacts with p53 homolog, ΔNp63α. This protein–protein interaction stabilizes ΔNp63α, through antagonizing function of the E3 ubiquitin ligase ITCH, inhibits nuclear translocation of ΔNp63α into the nucleus and suppresses ΔNp63α transactivation function. Additionally, we found that this functional crosstalk reverses cancer cells resistance to cisplatin, mediated by ΔNp63α, and consequently renders these cells more sensitive to undergo apoptosis. These findings suggest a functional crosstalk between WWOX and ΔNp63α in tumorigenesis.2013-11-19T14:30:30ZDrosophila Trap1 protects against mitochondrial dysfunction in a PINK1/parkin model of Parkinson's disease.Costa, A.C.Loh, S.H.Y.Martins, L. Miguelhttp://hdl.handle.net/2381/283992013-11-15T02:02:06Z2013-11-14T13:25:11ZTitle: Drosophila Trap1 protects against mitochondrial dysfunction in a PINK1/parkin model of Parkinson's disease.
Authors: Costa, A.C.; Loh, S.H.Y.; Martins, L. Miguel
Abstract: Mitochondrial dysfunction caused by protein aggregation has been shown to have an important role in neurological diseases, such as Parkinson's disease (PD). Mitochondria have evolved at least two levels of defence mechanisms that ensure their integrity and the viability of their host cell. First, molecular quality control, through the upregulation of mitochondrial chaperones and proteases, guarantees the clearance of damaged proteins. Second, organellar quality control ensures the clearance of defective mitochondria through their selective autophagy. Studies in Drosophila have highlighted mitochondrial dysfunction linked with the loss of the PTEN-induced putative kinase 1 (PINK1) as a mechanism of PD pathogenesis. The mitochondrial chaperone TNF receptor-associated protein 1 (TRAP1) was recently reported to be a cellular substrate for the PINK1 kinase. Here, we characterise Drosophila Trap1 null mutants and describe the genetic analysis of Trap1 function with Pink1 and parkin. We show that loss of Trap1 results in a decrease in mitochondrial function and increased sensitivity to stress, and that its upregulation in neurons of Pink1 mutant rescues mitochondrial impairment. Additionally, the expression of Trap1 was able to partially rescue mitochondrial impairment in parkin mutant flies; and conversely, expression of parkin rescued mitochondrial impairment in Trap1 mutants. We conclude that Trap1 works downstream of Pink1 and in parallel with parkin in Drosophila, and that enhancing its function may ameliorate mitochondrial dysfunction and rescue neurodegeneration in PD.
Description: PMCID: PMC35639932013-11-14T13:25:11ZHtrA2 deficiency causes mitochondrial uncoupling through the F₁F₀-ATP synthase and consequent ATP depletion.Plun-Favreau, H.Burchell, V.S.Holmström, K.M.Yao, Z.Deas, E.Cain, K.Fedele, V.Moisoi, NicoletaCampanella, M.Miguel Martins, L.Wood, N.W.Gourine, A.V.Abramov, A.Y.http://hdl.handle.net/2381/283962013-11-15T02:02:04Z2013-11-14T12:57:26ZTitle: HtrA2 deficiency causes mitochondrial uncoupling through the F₁F₀-ATP synthase and consequent ATP depletion.
Authors: Plun-Favreau, H.; Burchell, V.S.; Holmström, K.M.; Yao, Z.; Deas, E.; Cain, K.; Fedele, V.; Moisoi, Nicoleta; Campanella, M.; Miguel Martins, L.; Wood, N.W.; Gourine, A.V.; Abramov, A.Y.
Abstract: Loss of the mitochondrial protease HtrA2 (Omi) in mice leads to mitochondrial dysfunction, neurodegeneration and premature death, but the mechanism underlying this pathology remains unclear. Using primary cultures from wild-type and HtrA2-knockout mice, we find that HtrA2 deficiency significantly reduces mitochondrial membrane potential in a range of cell types. This depolarisation was found to result from mitochondrial uncoupling, as mitochondrial respiration was increased in HtrA2-deficient cells and respiratory control ratio was dramatically reduced. HtrA2-knockout cells exhibit increased proton translocation through the ATP synthase, in combination with decreased ATP production and truncation of the F1 α-subunit, suggesting the ATP synthase as the source of the proton leak. Uncoupling in the HtrA2-deficient mice is accompanied by altered breathing pattern and, on a cellular level, ATP depletion and vulnerability to chemical ischaemia. We propose that this vulnerability may ultimately cause the neurodegeneration observed in these mice.
Description: PMCID: PMC33882442013-11-14T12:57:26ZCell death in disease: from 2010 onwardsKnight, R.A.Melino, G.http://hdl.handle.net/2381/283952013-11-15T02:02:03Z2013-11-14T12:44:28ZTitle: Cell death in disease: from 2010 onwards
Authors: Knight, R.A.; Melino, G.
Abstract: The strong interest in cell death, and the shift in emphasis from basic mechanisms to translational aspects fostered the launch last year of the new sister journal of Cell Death and Differentiation, named Cell Death and Disease, to reflect its stronger focus towards clinical applications. Here, we review that first year of activity, which reflects an enthusiastic response by the scientific community. On the basis of this, we now launch two novel initiatives, the start of a new section dedicated to cancer metabolism and the opening of a new editorial office in Shanghai.2013-11-14T12:44:28Z